Summary: We will target at least seven BT agents identified as "Category A" by the Centers for Disease Control. The objectives are: (1) To evaluate existing PCR-based nucleic acid technology that rapidly detects BT pathogens to determine the feasibility of adapting them for the screening of blood. (2) To select PCR primers that will amplify diagnostic gene sequences from the pathogens. (3) To optimize methods for amplification of pathogen sequences from spiked blood samples. (4) To select oligonucleotide sequences corresponding to BT agent genes and print them on a microarray for hybridization to labeled, PCR amplified BT pathogen sequences and to sequences from closely related organisms that will be used as models to test the detection method. (5) To optimize methods for printing the oligonucleotides on glass slides. (6) To optimize methods for hybridization of the labeled PCR products to the microarray so that multiple suspected pathogens could be identified with one standard procedure. Description of Research: Selection of pathogens. The first rapid PCR detection techniques and prototype microarray are designed to detect agents identified by the CDC to have the greatest danger for use in bioterrorism (Bacillus anthracis, Yersinia pestis, Francisella tularensis, Venezuelan Equine Encephalitis Virus, Hemorrhagic viruses-Ebola and Marburg, and Variola major virus). We are focusing particular attention on pathogens with potential to contaminate the blood supply. We have selected a model organism related to each pathogen that can be used to test the system without the requirement for biosafety level 4 (BSL4) isolation procedures. Selection of amplicons. For each pathogen, one region of nucleic acid sequence has beeen selected for PCR amplification (the amplicon). The region has beeen selected primarily by searching the literature to identify nucleic acid sequences that have been amplified successfully with known primers (17 to 30 nucleotides in length). Amplicons have been selected that can discriminate the bioterror pathogens from other organisms and that have demonstrated sensitivity to less than 1000 organisms per milliler. Some of the targets already identified are the protective antigen (PA) gene, lethal factor (LF) gene, and edema factor (EF) gene for Bacillus anthracis; the plasminogen activator (PA) gene for Yersinia pestis, and the tul4 gene of Francisella tularensis to name a few. The quantitative PCR (qPCR) or "Taqman" approach will require a pair of primers that are 18-22 nucleotides in length, 40% to 60% GC content, melting temperature of 55 to 60oC, primers to be within 2oC melting temperature of each other, 1 to 2 nucleotide GC clamp, and amplicon size to be less than 200 base pairs in length. QPCR primers and probe have been synthesized for Bacillus anthracis. Selection of probes. For each amplicon, the qPCR method requires a fluorogenic probe that is 7 to 10oC higher than the primers' melting temperature, having no 5' terminal G residue, and its site to be less than 30 nucleotides from the corresponding strand primer. For the microarray, three internal oligonucleotides have been selected for each amplicon, 65-70 nucleotides in length, with melting temperatures 68-74?C. These probes have been searched against the Genbank database to insure they represent unique sequences. Oligonucleotide synthesis. Primers, probes and amplicons have been designed with the intent to minimize dimers, hairpins, long stretches of single base repeats, false priming sites; and to be able to multiplex the detection assay. Selected primer sequences were synthesized unmodified. Each probe was synthesized with an amino modification at the 5' end for covalently linking to an aldehyde coated glass slide. All oligos were column purified after synthesis to remove partial synthesis products. System development. The selected primers and probes will be combined into detection systems by two different approaches. I. Quantitative PCR. To achieve the shortest time for development of a usable test for the detection of BT agent nucleic acid in blood, primers and probes will be adapted for quantitative PCR. PCR will be performed on known amounts of template DNA or pathogen spiked into blood samples in a thermocycler. PCR products will be visualized on agarose gels. Sample preparation, cycling time and reaction buffer composition will be adjusted to reach maximum amplification. Subsequently, qPCR will be performed with the probes mentioned above in a Cephied Smartcycler (available at CBER). This device is equipped to measure fluorescence of up to 4 labeled probes. The sample preparation, reaction conditions and probe sequence will again be optimized to achieve the earliest possible cycle threshold (Ct) and satisfactory assay robustness (AR) with the minimum amount of pathogen in the sample. The earliest Ct and a good AR indicate maximum sensitivity measuring the correct target generated fluorescence distinct from background fluorescence. II. Microarrays. To achieve a more comprehensive, multiplex detection method, a microarray will be developed utilizing the same amplicons described above. A. Printing. The microarray probes for Leishmania, T. brucei, Hepatitis B Virus (HBV) and HCV as model organisms have been suspended in printing buffer in 384 well plates and spotted onto aldehyde coated glass slides with a robotic arrayer. Quality control oligos of "alien" sequence have been printed at the edges of each block. B. Sample preparation and amplification. Initial testing of PCR primers has been done with DNA samples and model organisms spiked into water. Detection of PCR products on an agarose gel stained with ethidium bromide was successful. Extraction procedures from whole blood samples will be optimized. Once successful PCR amplification was demonstrated, primer extension thermocyling (PET) reactions was performed to incorporate fluorescent label into single strand DNA for hybridization to the complementary strand probes on the microarray. Fluorescent-labeled DNA complementary to the quality control oligos was synthesized in similar fashion. C. Hybridization. Labeled PET products from the spiked samples and the quality control oligo were mixed with hybridization solution, and added to the microarray under a cover slip. The time (1-24 hours) and temperature (25?C-65?C) of incubation was optimized empirically. After hybridization, microarrays were washed to remove nonannealed DNA and dried for imaging. D. Microarray imaging. The hybridized microarrays were scanned and the image analyzed with the scanner software. The quality control spots were used to align the array grid over the image so that the identity of pathogen spots that show hybridization signal could be identified.